Biochemically stabilized HIV-1 Env trimer vaccine

ABSTRACT

Stabilized trimers of a clade A strain and a clade C strain of HIV-1 are provided. Broadly neutralizing antisera against HIV-1, methods of making broadly neutralizing antisera against HIV-1, broadly neutralizing vaccines against HIV-1, as well as methods of treating subjects infected with HIV, preventing HIV infection, and inhibiting HIV-mediated activities are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/082,601, filed on Apr. 8, 2011, which is a continuation of PCTApplication No. PCT/US2009/060494 designating the United States andfiled Oct. 13, 2009, which was published in the English language on Apr.15, 2010, under International Publication No. WO2010/042942; whichclaims the benefit of priority to U.S. Provisional Patent ApplicationNo. 61/104,449, filed on Oct. 10, 2008. Each disclosure is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under AI084794,AI078526, AI 066924, AI066305 and AI058727 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “689085.3U2 Sequence Listing” and a creation date of Apr. 4,2017, and having a size of 15 KB. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD

The present invention relates to novel methods and compositions forgenerating vaccines (e.g., HIV-1 vaccines).

BACKGROUND

Immunogens mimicking the trimeric structure of Envelope (Env) on thenative HIV-1 virion are actively being pursued as antibody-basedvaccines. However, it has proven difficult to produce biochemicallystable, immunogenic, trimeric Env immunogens.

SUMMARY

The present invention is based in part on the discovery of a stabilizedtrimer of a clade C strain of HIV-1 and the surprising discovery that astabilized trimer of a clade A strain and a stabilized trimer of a cladeC strain were each capable of eliciting a broadly neutralizing antibodyresponse in vivo.

Accordingly, in certain exemplary embodiments, a method oftherapeutically treating a subject infected with HIV, e.g., HIV-1,including contacting a subject infected with HIV with an isolatedpolypeptide comprising a stabilized trimer of an HIV envelopeglycoprotein, and producing neutralizing (e.g., broadly neutralizing)antisera in the subject to therapeutically treat the subject isprovided. In certain aspects, the method includes neutralizing HIV-1selected from one or more of clade A, clade B and clade C. In otheraspects, the method includes a gp140 trimer. In certain aspects, thegp140 trimer includes one or more variable loop peptides selected fromthe group consisting of V1, V2 and V3. In other aspects, the gp140trimer is derived from primary isolate CZA97.012 or 92UG037.8. Incertain aspects, HIV titer in the subject infected with HIV is decreasedafter contacting the subject with the isolated polypeptide.

In certain exemplary embodiments, a method of inhibiting an HIV-mediatedactivity in a subject in need thereof is provided. The method includescontacting an HIV-infected subject with an isolated polypeptideincluding a stabilized trimer of an HIV envelope glycoprotein, andproducing neutralizing (e.g., broadly neutralizing) antisera in thesubject to inhibit the HIV-mediated activity. In certain aspects, theHIV-mediated activity is viral spread. In other aspects, HIV titer inthe HIV-infected subject is decreased after contacting the subject withthe isolated polypeptide.

In certain exemplary embodiments, a method of preventing HIV infectionin a subject is provided. The method includes contacting a subject withan isolated polypeptide comprising a stabilized trimer of an HIVenvelope glycoprotein, and inducing in the subject immunity to HIV.

In certain exemplary embodiments, a vaccine is provided. The vaccineincludes a stabilized trimer comprising an isolated gp140 polypeptidederived from primary isolate CZA97.012 or primary isolate 92UG037.8 andhaving an oligomerization domain. The vaccine elicits production ofneutralizing (e.g., broadly neutralizing) antisera against HIV afterinjection into a subject.

In other exemplary embodiments, an isolated, antigenic, stabilizedtrimer of gp140 is provided. The stabilized trimer includes a gp140polypeptide derived from primary isolate CZA97.012 or primary isolate92UG037.8 and an oligomerization domain. The stabilized trimer elicitsproduction of neutralizing (e.g., broadly neutralizing) antisera againstHIV after injection into a subject.

In yet other exemplary embodiments, a vector encoding a polynucleotideincluding an antigenic, stabilized trimer of gp140 is provided. Thevector includes a gp140 polypeptide derived from primary isolateCZA97.012 or primary isolate 92UG037.8 and an oligomerization domain. Incertain aspects, the stabilized trimer of gp140 includes one or morevariable loop peptides selected from the group consisting of V1, V2 andV3. In other aspects, the stabilized trimer of gp140 comprises an aminoacid sequence having at least 85%, 90%, 95% or more sequence identity toSEQ ID NO:7 or SEQ ID NO:8. In other aspects, the stabilized trimer ofgp140 comprises an amino acid sequence including SEQ ID NO:7 or SEQ IDNO:8. In certain aspects, the stabilized trimer of gp140 elicitsproduction of broadly neutralizing antisera against HIV after injectioninto a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B graphically depict ELISA titers against gp140 in guinea pigsera. Sera obtained 4 weeks after each immunization were tested inendpoint ELISAs against the clade A and clade C trimers in the (FIG. 1A)clade A and (FIG. 1B) clade C vaccinated guinea pigs. Graphs showgeometric mean titers for each time point+/−standard deviation.Horizontal line indicates back ground threshold.

FIGS. 2A-2B graphically depict summaries of neutralizing antibody (NAb)titers against tier 2 viruses. NAb titers against six clade A (red),clade B (blue), and clade C (green) tier 2 primary isolates aresummarized for each guinea pig. (FIG. 2A) pre- and (FIG. 2B)post-immunization. Horizontal line indicates titer>60 cut-off forpositivity.

FIGS. 3A-3B graphically depict sample raw neutralization data withpurified IgG against the tier 2 clade C virus ZM109F.PB4. Serialdilutions of purified IgG from (FIG. 3A) clade A or (FIG. 3B) clade Ctrimer immunized guinea pigs and naive control animals were tested forNAb activity against the tier 2 clade C virus ZM109F.PB4.

FIGS. 4A-4D depict antibody responses to variable loop peptides. Pre-and post-immunization sera from (FIG. 4A) clade A and (FIG. 4B) clade Ctrimer immunized animals were assessed by ELISA against homologous andheterologous V1, V2 and V3 loop peptides. Graph depicts geometric meantiters for each group+/−standard deviation. Horizontal line indicatesbackground threshold. (FIG. 4C) Purified IgG obtained from the sera ofrepresentative animals immunized with the clade A (guinea pig #5) andclade C (guinea pig #10) trimers were pre-incubated with linear V3 loopor scrambled peptides and then tested in the TZM.b1 neutralizationassays against SF162.LS (tier 1-B), ZM109F.PB4 (tier 2-C) and 6535.3(Tier 2-B) viruses. (FIG. 4D) Sequence alignment of linear 92UG037.8(SEQ ID NOs:3, 5 and 7) and CZA97012 (SEQ ID NOs:4, 6 and 8) V1-V3peptide loops. Amino acid residues highlighted in greyscale indicatehomology between sequences at that position.

FIGS. 5A-5D graphically depict ELISA titers against gp140 followingheterologous prime/boost vaccination regimens. Sera obtained 4 weeksafter each immunization in the DNA/protein boost groups (FIG. 5A, FIG.5B) and the DNA/rAd26 groups (FIG. 5C, FIG. 5D) groups were assessed byELISAs. Graphs show geometric mean titers for each time point+/−standarddeviation. 3* indicates ELISA titers after 3 immunizations but prior toboosting. Horizontal line indicates background threshold.

FIG. 6 depicts HIV-1 tier 1 and 2 neutralization titers of guinea pigsera in TZM-b1 assays. Pre-immunization (Pre) and post 3^(rd) trimervaccination (Post) sera were tested against panels of tier 1 and tier 2clade A, B and C pseudoviruses in TZM.b1 neutralization assays. Valuesshown are the serum dilutions representing the ID50 titers for eachanimal. Values highlighted in yellow indicate positive responses definedas: (i) >3-fold above pre-immune background (ii) >2-fold above a murineleukemia virus (MuLv) control, and (iii) absolute ID₅₀ titer>60.

FIG. 7 depicts a summary of NAb titers against tier 2 clade A, B and Cviruses. The number and percent of positive samples tested are shown.

FIG. 8 depicts ID₅₀ neutralization of select tier 1 and 2 isolates withpurified guinea pig IgG. IgG purified from individual guinea pig wastested in TZM.b1 neutralization assays against a select panel of tier 1and 2 viruses. Data are represented as IC₅₀ titers in μg/ml (lowernumbers reflect better neutralization). Values highlighted in yellowindicate samples with positive neutralizing activity.

FIG. 9 depicts HIV-1 tier 1 neutralization titers of guinea pig sera inTZM-b1 assays following prime/boost vaccination regimens.Pre-immunization, post-third DNA vaccination, and post rAd26/proteinboost sera from guinea pigs were tested for NAb responses against tier 1viruses in TZM.b1 neutralization assays. Values shown are the serumdilutions representing the ID50 titers for each animal. Highlightedvalues indicate positive responses.

FIG. 10 depicts a surface plasminogen resonance assay of92UG037.8-gp140-Fd binding to CD4 (left panel, K_(d) 1.9 nM), and to2G12 (right panel, K_(d)=17.9 nM).

FIG. 11 depicts a surface plasminogen resonance assay of92UG037.8-gp140-Fd binding to mAb 17b (left panel), and to Cluster ImAbs (right panel).

FIGS. 12A-12B depicts the amino acid sequences of the 92UG037.8-gp140-6×His trimer (FIG. 12A) (SEQ ID NO:1) and the CZA97.012-gp140-6× Histrimer (FIG. 12B) (SEQ ID NO:2).

FIG. 13 schematically depicts foldon-stabilized HIV-1 gp140 trimers(gp140-Fd). Gp160 is the full-length precursor. Various segments ofgp120 and gp41 are designated as follows: C1-C5, conserved regions 1-5;V1-V5, variable regions 1-5; F, fusion peptide; HR1, heptad repeat 1;C-C loop, the immunodominant loop with a conserved disulfide bond; HR2,heptad repeat 2; MPER, membrane proximal external region; TM,transmembrane anchor; CT, cytoplasmic tail. Gp140-Fd represents theuncleaved ectodomain of gp160 with a T4 fibritin foldon trimerizationtag and His tag at its C-terminus.

DETAILED DESCRIPTION

Most antibodies induced by HIV-1 are ineffective at preventinginitiation or spread of infection, as they are either non-neutralizingor narrowly isolate-specific. One of the biggest challenges in HIVvaccine development is to design an HIV envelope immunogen that caninduce protective, neutralizing antibodies effective against the diverseHIV-1 strains that characterize the global pandemic. Indeed, thegeneration of “broadly neutralizing” antibodies that recognizerelatively conserved regions on the envelope glycoprotein are rare. Thepresent invention is based in part on the discovery of stabilizedtrimeric HIV-1 envelope proteins that surprisingly elicit a broadlyneutralizing antibody response in vivo.

In certain exemplary embodiments, the compounds and methods describedherein are used to inhibit or decrease infectivity of one or morepathogens (e.g., viruses, bacteria, fungi, parasites and the like) thathave one or more multimeric surface proteins. Accordingly, the presentinvention is directed in part to stabilized oligomer (e.g., trimer)conformations of the envelope protein (e.g., gp41) of a humanimmunodeficiency virus (e.g., HIV-1) and methods for their use. Incertain aspects, the compounds and methods described herein are used toinhibit or decrease one or more HIV-mediated activities (e.g.,infection, fusion (e.g., target cell entry and/or syncytia formation),viral spread and the like) in a subject, which can, in turn, decreaseHIV titer.

As used herein, the terms “inhibiting” or “decreasing” with respect toHIV refer to an inhibition or decrease of an HIV-mediated activity(e.g., infection, fusion (e.g., target cell entry and/or syncytiaformation), viral spread and the like) and/or a decrease in viral titer.For example, an HIV-mediated activity may be decreased by 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more.

HIV is a member of the genus Lentivirinae, part of the family ofRetroviridae. Two species of HIV infect humans: HIV-1 and HIV-2. As usedherein, the terms “human immunodeficiency virus” and “HIV” refer, butare not limited to, HIV-1 and HIV-2. In certain exemplary embodiments,the envelope proteins described herein refer to those present on any ofthe five serogroups of lentiviruses that are recognized: primate (e.g.,HIV-1, HIV-2, simian immunodeficiency virus (SIV)); sheep and goat(e.g., visna virus, caprine arthritis encephalitis virus); horse (equineinfectious anemia virus); cat (e.g., feline immunodeficiency virus(FIV)); and cattle (e.g., bovine immunodeficiency virus (BIV)) (SeeInternational Committee on Taxonomy of Viruses descriptions).

HIV is categorized into multiple clades with a high degree of geneticdivergence. As used herein, the term “clade” refers to related humanimmunodeficiency viruses classified according to their degree of geneticsimilarity. There are currently three groups of HIV-1 isolates: M, N andO. Group M (major strains) consists of at least ten clades, A through J.Group O (outer strains) may consist of a similar number of clades. GroupN is a new HIV-1 isolate that has not been categorized in either group Mor O. In certain exemplary embodiments, a broadly neutralizing antibodydescribed herein will recognize and raise an immune response againsttwo, three, four, five, six, seven, eight, nine, ten or more cladesand/or two or more groups of HIV.

As used herein, the term “envelope glycoprotein” refers, but is notlimited to, the glycoprotein that is expressed on the surface of theenvelope of HIV virions and the surface of the plasma membrane of HIVinfected cells. The env gene encodes gp160, which is proteolyticallycleaved into gp120 and gp140. Gp120 binds to the CD4 receptor on atarget cell that has such a receptor, such as, e.g., a T-helper cell.Gp41 is non-covalently bound to gp120, and provides the second step bywhich HIV enters the cell. It is originally buried within the viralenvelope, but when gp120 binds to a CD4 receptor, gp120 changes itsconformation causing gp41 to become exposed, where it can assist infusion with the host cell.

As used herein, the term “oligomer,” when used in the context of aprotein and/or polypeptide is intended to include, but is not limitedto, a protein or polypeptide having at least two subunits. Oligomersinclude, but are not limited to, dimers, trimers, tetramers, pentamers,hexamers, heptamers, octamers, nonamers, decamers and the like.

As used herein, the term “stabilized oligomer” refers, but is notlimited to, an oligomer that includes a protein and/or polypeptidesequence that increases the stability (e.g., via the presence of one ormore oligomerization domains) of the oligomeric structure (e.g., reducesthe ability of the oligomer to dissociate into monomeric units). Incertain exemplary embodiments, a stabilized oligomer is a stabilizedtrimer.

As used herein, the term “oligomerization domain” refers, but is notlimited to, a polypeptide sequence that can be used to increase thestability of an oligomeric envelope protein such as, e.g., to increasethe stability of an HIV gp41 trimer. Oligomerization domains can be usedto increase the stability of homooligomeric polypeptides as well asheterooligomeric polypeptides. Oligomerization domains are well known inthe art.

As used herein, the terms “trimerization domain” and “trimerization tag”refer to an oligomerization domain that stabilizes trimeric polypeptides(e.g., a gp41 homotrimeric polypeptide). Examples of trimerizationdomain include, but are not limited to, the T4-fibritin “foldon” trimer;the coiled-coil trimer derived from GCN4 (Yang et al. (2002) J Virol.76:4634); the catalytic subunit of E. coli aspartate transcarbamoylaseas a trimer tag (Chen et al. (2004) J Virol. 78:4508). Trimerizationdomains are well known in the art.

As used herein, the term “protein tag” refers, but is not limited to, apolypeptide sequence that can be added to another polypeptide sequencefor a variety of purposes. In certain exemplary embodiments, a proteintag may be removed from a larger polypeptide sequence when it is nolonger needed. Protein tags include, but are not limited to, affinitytags (e.g., poly-His tags, chitin binding protein (CBP), maltose bindingprotein (MBP), glutathione-s-transferase (GST) and the like),solubilization tags (e.g., include thioredoxin (TRX), poly(NANP) MBP,GST and the like), chromatography tags (e.g., polyanionic amino acidssuch as the FLAG epitope), epitope tags (e.g., FLAG-tag, V5-tag,c-myc-tag, HA-tag and the like), fluorescent tags (e.g., greenfluorescent protein (GFP), yellow fluorescent protein (YFP), cyanfluorescence protein (CFP) and the like), bioluminescent tags (e.g.,luciferase (e.g., bacterial, firefly, click beetle, sea pansy (Renilla)and the like), luciferin, aequorin and the like), enzyme modificationtags (e.g., biotin ligase and the like) and the like. Protein tags arewell known in the art and their reagents are often commerciallyavailable.

In certain exemplary embodiments, a stabilized trimer of an envelopeglycoprotein described herein can be administered to a subject in whomit is desirable to promote an immune response. In other exemplaryembodiments, a nucleic acid sequence encoding one or more stabilizedtrimers of an envelope protein described herein can be administered to asubject in whom it is desirable to promote an immune response.

Accordingly, one or more stabilized oligomers (e.g., stabilized trimers)described herein can be used as immunogens to produce anti-oligomer(e.g., anti-trimer) antibodies in a subject, to inhibit or preventinfection by HIV and/or to inhibit or prevent the spread of HIV in aninfected individual. One or more stabilized oligomers (e.g., stabilizedtrimers) of an envelope glycoprotein described herein can be used as animmunogen to generate antibodies that bind wild-type envelopeglycoprotein (i.e., gp41 and/or gp160) using standard techniques forpolyclonal and monoclonal antibody preparation.

In certain exemplary embodiments, a stabilized oligomer (e.g.,stabilized trimer) of an envelope glycoprotein is capable of eliciting abroadly neutralizing antibody response in a host. As used herein, theterms “neutralizing antibody response” and “broadly neutralizingantibody response” are well known in the art and refer to the ability ofone or more antibodies to react with an infectious agent to destroy orgreatly reduce the virulence of the infectious agent. The presence ofsuch a response has the potential to prevent the establishment ofinfection and/or to significantly reduce the number of cells that becomeinfected with HIV, potentially delaying viral spread and allowing for abetter control of viral replication in the infected host. A broadlyneutralizing antibody against HIV will typically bind a variety ofdifferent clades, groups or mutants of HIV.

As used herein, the term “immune response” is intended to include, butis not limited to, T and/or B cell responses, that is, cellular and/orhumoral immune responses. The immune response of a subject can bedetermined by, for example, assaying antibody production, immune cellproliferation, the release of cytokines, the expression of cell surfacemarkers, cytotoxicity, and the like. As used herein, the term “immunecell” is intended to include, but is not limited to, cells that are ofhematopoietic origin and play a role in an immune response. Immune cellsinclude, but are not limited to, lymphocytes, such as B cells and Tcells; natural killer cells; myeloid cells, such as monocytes,macrophages, eosinophils, mast cells, basophils, and granulocytes.

A stabilized oligomer (e.g., trimer) of an envelope glycoproteintypically is used to prepare antibodies by immunizing a suitablesubject, (e.g., rabbit, guinea pig, goat, mouse or other mammal) withthe immunogen. An appropriate immunogenic preparation can contain, forexample, a recombinantly expressed stabilized trimer of an envelopeglycoprotein or a chemically synthesized stabilized trimer of anenvelope glycoprotein. The preparation can further include an adjuvant,such as Freund's complete or incomplete adjuvant, Ribi adjuvant, orsimilar immunostimulatory agent. Immunization of a suitable subject withan immunogenic stabilized oligomer (e.g., trimer) of an envelopeglycoprotein preparation induces a polyclonal anti-envelope (e.g.,anti-gp41 and/or anti-gp160) antibody response, e.g., an anti-HIVantibody response.

Accordingly, in certain exemplary embodiments, anti-stabilized gp41trimer antibodies are provided. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as the envelope glycoprotein (e.g., gp41 and/or gp160). Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin. In certain embodiments,polyclonal and/or monoclonal antibodies that bind the envelopeglycoprotein (e.g., gp41 and/or gp160) are provided. The term“monoclonal antibody” or “monoclonal antibody composition,” as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of the envelope glycoprotein (e.g., gp41 and/orgp160). A monoclonal antibody composition thus typically displays asingle binding affinity for a particular the envelope glycoprotein(e.g., gp41 and/or gp160) with which it immunoreacts.

Polyclonal anti-stabilized trimer envelope glycoprotein (e.g., gp41and/or gp160) antibodies can be prepared as described above byimmunizing a suitable subject with a stabilized oligomer (e.g., trimer)of an envelope glycoprotein immunogen as described herein. Theanti-stabilized trimer of an envelope glycoprotein antibody titer in theimmunized subject can be monitored over time by standard techniques,such as with an enzyme linked immunosorbent assay (ELISA) usingimmobilized gp41. If desired, the antibody molecules directed againstgp41 can be isolated from the mammal (e.g., from the blood) and furtherpurified by well known techniques, such as protein A chromatography toobtain the IgG fraction. At an appropriate time after immunization,e.g., when the anti-gp41 antibody titers are highest, antibody-producingcells can be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975) Nature 256:495-497)(see also, Brown et al. (1981) J Immunol. 127:539-46; Brown et al.(1980) J Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad.Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J Cancer 29:269-75), thehuman B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today4:72), the EBV-hybridoma technique (Cole et al. (1985), MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triomatechniques. The technology for producing monoclonal antibody hybridomasis well known (see generally R. H. Kenneth, in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); E. A. Lerner (1981) Yale J Biol. Med. 54:387-402; Gefter etal. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a stabilized trimer of an envelopeglycoprotein immunogen as described above, and the culture supernatantsof the resulting hybridoma cells are screened to identify a hybridomaproducing a monoclonal antibody that binds gp41.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-stabilized trimer of an envelope glycoprotein monoclonal antibody(see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.Somatic Cell Genet., cited supra; Lerner, Yale J. Biol Med. (supra);Kenneth, Monoclonal Antibodies, (supra)). Moreover, the ordinarilyskilled worker will appreciate that there are many variations of suchmethods which also would be useful. Typically, the immortal cell line(e.g., a myeloma cell line) is derived from the same mammalian speciesas the lymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line. Particularlysuitable immortal cell lines are mouse myeloma cell lines that aresensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/0-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of astabilized trimer of an envelope glycoprotein are detected by screeningthe hybridoma culture supernatants for antibodies that bind gp41, e.g.,using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-stabilized trimer of an envelope glycoprotein antibodycan be identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with agp41 protein to thereby isolate immunoglobulin library members that bindgp41. Kits for generating and screening phage display libraries arecommercially available (e.g., Recombinant Phage Antibody System, Pfizer,New York, N.Y.; and the SURFZAP™ Phage Display Kit, Stratagene, LaJolla, Calif.). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT International Publication No. WO92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/20791; Markland et al.PCT International Publication No. WO92/15679; Breitling et al. PCTInternational Publication WO93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl Acad Sci USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucl. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

Additionally, recombinant anti-stabilized trimer of an envelopeglycoprotein antibodies, such as chimeric and humanized monoclonalantibodies, comprising both human and non-human portions, which can bemade using standard recombinant DNA techniques, are within the scope ofthe invention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J Immunol.141:4053-4060.

In certain exemplary embodiments, compositions and methods for enhancingthe immune response of a subject to a human immunodeficiency virus areprovided. As used herein, the terms “subject” and “host” are intended toinclude living organisms such as mammals. Examples of subjects and hostsinclude, but are not limited to, horses, cows, sheep, pigs, goats, dogs,cats, rabbits, guinea pigs, rats, mice, gerbils, non-human primates(e.g., macaques), humans and the like, non-mammals, including, e.g.,non-mammalian vertebrates, such as birds (e.g., chickens or ducks) fishor frogs (e.g., Xenopus), and non-mammalian invertebrates, as well astransgenic species thereof.

In certain exemplary embodiments, vectors such as, for example,expression vectors, containing a nucleic acid encoding one or morestabilized trimers of an envelope protein described herein are provided.As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably. However, the invention is intendedto include such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

In certain exemplary embodiments, the recombinant expression vectorscomprise a nucleic acid sequence (e.g., a nucleic acid sequence encodingone or more stabilized trimers of an envelope protein described herein)in a form suitable for expression of the nucleic acid sequence in a hostcell, which means that the recombinant expression vectors include one ormore regulatory sequences, selected on the basis of the host cells to beused for expression, which is operatively linked to the nucleic acidsequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequenceencoding one or more stabilized trimers of an envelope protein is linkedto the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like. The expression vectors described herein can beintroduced into host cells to thereby produce proteins or portionsthereof, including fusion proteins or portions thereof, encoded bynucleic acids as described herein (e.g., one or more stabilized trimersof an envelope protein).

In certain exemplary embodiments, nucleic acid molecules describedherein can be inserted into vectors and used as gene therapy vectors.Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see, e.g., U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA. 91:3054). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, adeno-associated virus vectors, and the like,the pharmaceutical preparation can include one or more cells whichproduce the gene delivery system (See Gardlik et al. (2005) Med. Sci.Mon. 11:110; Salmons and Gunsberg (1993) Hu. Gene Ther. 4:129; and Wanget al. (2005) J Virol. 79:10999 for reviews of gene therapy vectors).

Recombinant expression vectors of the invention can be designed forexpression of one or more encoding one or more stabilized trimers of anenvelope protein in prokaryotic or eukaryotic cells. For example, one ormore vectors encoding one or more stabilized trimers of an envelopeprotein can be expressed in bacterial cells such as E. coli, insectcells (e.g., using baculovirus expression vectors), yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40); pMAL (New EnglandBiolabs, Beverly, Mass.); and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector encoding one or morestabilized trimers of an envelope protein is a yeast expression vector.Examples of vectors for expression in yeast S. cerevisiae includepYepSec1 (Baldari, et. al., (1987) EMBO J 6:229-234); pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943); pJRY88 (Schultz et al., (1987) Gene54:113-123); pYES2 (Invitrogen Corporation, San Diego, Calif.); and picZ(Invitrogen Corporation).

Alternatively, one or more stabilized trimers of an envelope protein canbe expressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In certain exemplary embodiments, a nucleic acid described herein isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, adenovirus 2, cytomegalovirusand simian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In certain exemplary embodiments, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include lymphoid-specificpromoters (Calame and Eaton (1988) Adv. Immunol 43:235), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J 8:729)and immunoglobulins (Banetji et al. (1983) Cell 33:729; Queen andBaltimore (1983) Cell 33:741), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA. 86:5473), pancreas-specific promoters (Edlund et al. (1985) Science230:912), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Developmentally-regulated promoters are also encompassed,for example the murine hox promoters (Kessel and Gruss (1990) Science249:374) and the a-fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537).

In certain exemplary embodiments, host cells into which a recombinantexpression vector of the invention has been introduced are provided. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, oneor more stabilized trimers of an envelope protein can be expressed inbacterial cells such as E. coli, viral cells such as retroviral cells,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Delivery of nucleic acids described herein (e.g., vector DNA) can be byany suitable method in the art. For example, delivery may be byinjection, gene gun, by application of the nucleic acid in a gel, oil,or cream, by electroporation, using lipid-based transfection reagents,or by any other suitable transfection method.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection (e.g., usingcommercially available reagents such as, for example, LIPOFECTIN®(Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE® (Invitrogen),FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI®(Polyplus-transfection Inc., New York, N.Y.), EFFECTENE® (Qiagen,Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), orelectroporation (e.g., in vivo electroporation). Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring harborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

Embodiments of the invention are directed to a first nucleic acid (e.g.,a nucleic acid sequence encoding one or more stabilized trimers of anenvelope glycoprotein) or polypeptide sequence (e.g., one or morestabilized trimers of an envelope glycoprotein) having a certainsequence identity or percent homology to a second nucleic acid orpolypeptide sequence, respectively. Techniques for determining nucleicacid and amino acid “sequence identity” are known in the art. Typically,such techniques include determining the nucleotide sequence of genomicDNA, mRNA or cDNA made from an mRNA for a gene and/or determining theamino acid sequence that it encodes, and comparing one or both of thesesequences to a second nucleotide or amino acid sequence, as appropriate.In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Two or more sequences(polynucleotide or amino acid) can be compared by determining their“percent identity.” The percent identity of two sequences, whethernucleic acid or amino acid sequences, is the number of exact matchesbetween two aligned sequences divided by the length of the shortersequences and multiplied by 100.

An approximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745. Anexemplary implementation of this algorithm to determine percent identityof a sequence is provided by the Genetics Computer Group (Madison, Wis.)in the “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.).

One method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages, the Smith-Waterman algorithm canbe employed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at theNCBI/NLM web site.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions that form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNAsequences, or two polypeptide sequences are “substantially homologous”to each other when the sequences exhibit at least about 80%-85%, atleast about 85%-90%, at least about 90%-95%, or at least about 95%-98%sequence identity over a defined length of the molecules, as determinedusing the methods above. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified DNA orpolypeptide sequence. DNA sequences that are substantially homologouscan be identified in a Southern hybridization experiment under, forexample, stringent conditions, as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.; Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supra).Such assays can be conducted using varying degrees of selectivity, forexample, using conditions varying from low to high stringency. Ifconditions of low stringency are employed, the absence of non-specificbinding can be assessed using a secondary probe that lacks even apartial degree of sequence identity (for example, a probe having lessthan about 30% sequence identity with the target molecule), such that,in the absence of non-specific binding events, the secondary probe willnot hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” conditionstypically hybridizes under conditions that allow detection of a targetnucleic acid sequence of at least about 10-14 nucleotides in lengthhaving at least approximately 70% sequence identity with the sequence ofthe selected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization, supra).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook et al., supra).

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% identical to each othertypically remain hybridized to each other. In one aspect, the conditionsare such that sequences at least about 70%, at least about 80%, at leastabout 85% or 90% or more identical to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A non-limitingexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C., at 55° C., or at 60° C. or65° C.

A first polynucleotide is “derived from” a second polynucleotide if ithas the same or substantially the same base-pair sequence as a region ofthe second polynucleotide, its cDNA, complements thereof, or if itdisplays sequence identity as described above. A first polypeptide isderived from a second polypeptide if it is encoded by a firstpolynucleotide derived from a second polynucleotide, or displayssequence identity to the second polypeptides as described above. In thepresent invention, when a gp41 protein is “derived from HIV” the gp41protein need not be explicitly produced by the virus itself, the virusis simply considered to be the original source of the gp41 proteinand/or nucleic acid sequences that encode it. Gp41 proteins can, forexample, be produced recombinantly or synthetically, by methods known inthe art, or alternatively, gp41 proteins may be purified fromHIV-infected cell cultures.

In certain exemplary embodiments, one or more antibodies, one or morestabilized trimers of an envelope protein and/or nucleic acid sequencesencoding one or more stabilized trimers of an envelope protein describedherein can be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule or protein and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

In certain exemplary embodiments, a pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating one ormore antibodies, one or more stabilized trimers of an envelope proteinand/or nucleic acid sequences encoding one or more stabilized trimers ofan envelope protein described herein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: A binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic, acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant: such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, one or more antibodies, one or more stabilizedtrimers of an envelope protein and/or nucleic acid sequences encodingone or more stabilized trimers of an envelope protein described hereinare prepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These may beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Nasal compositions generally include nasal sprays and inhalants. Nasalsprays and inhalants can contain one or more active components andexcipients such as preservatives, viscosity modifiers, emulsifiers,buffering agents and the like. Nasal sprays may be applied to the nasalcavity for local and/or systemic use. Nasal sprays may be dispensed by anon-pressurized dispenser suitable for delivery of a metered dose of theactive component. Nasal inhalants are intended for delivery to the lungsby oral inhalation for local and/or systemic use. Nasal inhalants may bedispensed by a closed container system for delivery of a metered dose ofone or more active components.

In one embodiment, nasal inhalants are used with an aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A non-aqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers maybe used to minimize exposing the agent to shear, which can result indegradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

One or more antibodies, one or more stabilized trimers of an envelopeprotein and/or one or more nucleic acid sequences encoding one or morestabilized trimers of an envelope protein described herein can also beprepared in the form of suppositories (e.g., with conventionalsuppository bases such as cocoa butter and other glycerides) orretention enemas for rectal delivery.

In one embodiment, one or more antibodies, one or more stabilizedtrimers of an envelope protein and/or one or more nucleic acid sequencesencoding one or more stabilized trimers of an envelope protein describedherein are prepared with carriers that will protect them against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of one or more antibodies, one or morestabilized trimers of an envelope protein and/or one or more nucleicacid sequences encoding one or more stabilized trimers of an envelopeprotein described herein can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50. Compounds whichexhibit large therapeutic indices are preferred. While compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue in order to minimize potential damage to uninfected cells and,thereby, reduce side effects.

Data obtained from cell culture assays and/or animal studies can be usedin formulating a range of dosage for use in humans. The dosage typicallywill lie within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In certain exemplary embodiments, a method for treatment of a viralinfection, e.g., HIV infection, includes the step of administering atherapeutically effective amount of an agent (e.g., one or moreantibodies, one or more stabilized trimers of an envelope protein, anucleic acid sequence that encodes one or more stabilized trimers of anenvelope protein and the like) which modulates (e.g., inhibits), one ormore envelope protein (e.g., gp41) activities (e.g., mediating viralfusion (e.g., viral entry and/or syncytia formation)) to a subject. Asdefined herein, a therapeutically effective amount of agent (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight, fromabout 0.01 to 25 mg/kg body weight, from about 0.1 to 20 mg/kg bodyweight, or from about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciatethat certain factors may influence the dosage required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of an inhibitor caninclude a single treatment or, in certain exemplary embodiments, caninclude a series of treatments. It will also be appreciated that theeffective dosage of inhibitor used for treatment may increase ordecrease over the course of a particular treatment. Changes in dosagemay result from the results of diagnostic assays as described herein.The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. This example is not to be construed as limiting thescope of the invention as these and other equivalent embodiments will beapparent in view of the present disclosure, figures and accompanyingclaims.

Example I Neutralization Capacity of Biochemically Stable HIV-1 gp140Trimers in a Guinea Pig Model

Preclinical evaluation of candidate Env immunogens is critical forconcept testing and for prioritization of vaccine candidates.Luciferase-based virus neutralization assays in TZM.b1 cells (Li et al.(2005) J Virol. 79:10108; Montefiori (2005) Curr. Prot. Immunol. Chapter12:Unit 1211) have been developed as high throughput assay that can bestandardized (Montefiori (2009) Methods Mol. Biol. 485:395; Polonis etal. (2008) Virology 375:315). However, optimal use of this assayrequired the generation of standardized virus panels derived frommultiple clades and reflecting both easy-to-neutralize (tier 1) andprimary isolates (tier 2) viruses (Li et al., Supra).

By screening a panel of primary HIV-1 isolates, two viruses, CZA97.012(Clade C) (Rodenburg et al. (2001) AIDS Res. Hum. Retroviruses 17:161)and 92UG037.8 (Clade A) (Chen et al., Supra) were identified thatyielded biochemically homogenous and stable trimers with well-definedand uniform antigenic properties (Burke et al. (2009) Virology 387:147).The addition of the T4 bacteriophage fibritin “fold-on” (Fd)trimerization domain further increased their yield and purity (Frey etal. (2008) Proc. Natl. Acad. Sci. USA 105:3739). As described furtherherein, the immunogenicity of these stabilized clade A and clade C gp140trimers was assessed in guinea pigs using a panel of tier 1 and tier 2isolates from clades A, Band C.

Production of Stable, Homogenous HIV-1 gp140 Trimers

Env gp140 trimers derived from primary isolates 92UG037.8 (clade A) andCZA97.012 (clade C) were stabilized with a T4-fibritin “foldon”C-terminal trimerization tag (FIG. 13) and produced in T.ni (High 5)cells. The biochemical purity and stability was determined as follows:Purified CZA97.012 (clade C) gp140 trimer was resolved by gel-filtrationchromatography on Superpose 6 columns. The apparent molecular mass wascalculated by using standards thryoglobulin (670 kDa), ferritin (440kDa), and catalase (232 kDa). Peak fractions were pooled and analyzed bySDS-PAGE. The clade C trimer was treated with various concentrations (0,0.05, 0.25, 0.5, 1, 2, 5 mM) of ethylene glycolbis(succinimidylsuccinate). Cross-linked products were analyzed bySDS-PAGE using a 4% gel. The molecular standard was cross-linkedphosphorylase b (Sigma). Similar analyses have been previously reportedfor the purified clade A 92UG037.8 gp140 trimer (Frey et al., Supra).The CZA97.012 (clade C) trimer showed similar purity and homogeneity asdetermined by size exclusion chromatography and chemical cross linkingthat confirmed that it was monodisperse and trimeric.

A luciferase reporter gene assay was performed in TZM-b1 cells (agenetically engineered cell line that expresses CD4, CXCR4 and CCRS andcontains Tat-inducible Luc and Gal reporter genes) based on single roundinfection with molecularly cloned Env-pseudotyped viruses. This assayresulted in a high success rate in single round infections, increasedassay capacity (e.g., a two day assay), increased precision (e.g.,accurately measured 50% neutralization), and an improved level ofstandardization (e.g., a stable cell line). The luciferase reporter geneassay was optimized and validated.

Binding Antibody Responses Elicited by Clade A and Clade C Trimers

A multi-tiered approach was used to assess vaccine-elicited neutralizingantibody responses. In Tier 1, vaccine strain(s) andneutralization-sensitive strains were not included in the vaccine. InTier 2, a panel of heterologous viruses matching the genetic subtype(s)of the vaccine (e.g., 12 viruses per panel) was used. This tier couldoptionally include additional strains from vaccine trial sites. In Tier3, a multi-clade panel comprised of six Tier 2 viruses evaluated in Tier2 was used. Tier 3 could optionally include additional strains from anoptional vaccine trial site.

In a preliminary study, the immunogenicity of a 92UG037.8 (clade A)gp120 monomer core immunogen was assessed in guinea pigs, which elicitedonly minimal neutralizing antibody (Nab) responses against tier 1neutralization sensitive viruses. The immunogenicity of 92UG037.8 (cladeA) and CZA97.012 (clade C) trimers that were selected and engineered formaximum stability and conformational homogeneity were then focused on.These immunogens contained the gp140 sequence fused to the T4 fibritinfold-on trimerization domain.

Preliminary 92UG037.8 gp120 monomer experiments were performed asfollows. A monomeric 92UG037.8-gp120 ‘core’ that was devoid of V1-V3regions and had amino- and carboxy-terminal truncations was generated.Guinea pigs were immunized at four week intervals with 100 μg 92UG037.8gp120 monomer in Ribi adjuvant. Sera were obtained four weeks after eachimmunization, and were tested against the 92UG037.8 gp120 antigen in anend-point ELISA assay.

Env gp140 trimers derived from primary isolates 92UG037.8 (clade A) andCZA97.012 (clade C) were stabilized with a T4-fibritin foldon C-terminaltrimerization tag and produced in T.ni cells (HIGH FIVE™, Invitrogen,Carlsbad, Calif.). Initial biochemical analyses were performed bysize-exclusion chromatography and chemical cross-linking. 92UG037.8(clade A) and CZA97.012 (clade C) gp140-Fd trimers were purified tohomogeneity and exhibited single peaks as determined by size-exclusionchromatography. Expected molecular weights and oligomerization stateswere observed for both 92UG037.8 and CZA97.012 trimers by Coomassiestaining and chemical cross-linking, respectively. The sedimentationequilibrium of 92UG037.8-gp140-Fd and its in vitro cleavage by humanplasmin were determined. The molecular mass was determined to be409+/−10 kDa (confirming the expected value of approximately 405 kDa).

92UG037.8-gp140-Fd was able to bind both soluble CD4 and monoclonalantibody (mAb) 2G12 (a broadly neutralizing mAb that recognizescarbohydrates on the outer gp120 surface). 92UG037.8-gp140-Fd also boundCD4i mAb 17b (a monoclonal CD4-induced (CD4i) neutralizing antibody thathas a binding epitope which partially overlaps with the co-receptor CCR5binding site of gp120) and cluster I mAbs (mAbs that arenon-neutralizing and react with the immunodominant region of gp41 (aminoacids 579-613); Cluster II mAbs react with MPER gp41 amino acids 644-667and are either non-neutralizing or neutralizing (e.g., mAbs 2F5, 4E10,Z13)).

Guinea pigs (n=5/group) were immunized with 100 μg of the clade A orclade C gp140 trimer in Ribi adjuvant s.q./i.p. at weeks 0, 5 and 10.Serum was obtained 4 weeks after each immunization. Env-specific bindingantibodies were assessed by endpoint ELISAs to both clade A and clade Cgp140. High titer binding antibody responses were observed in both cladeA and clade C trimer immunized guinea pigs. Responses were detectedafter a single immunization and increased to a mean 6.5 log titerfollowing the second and third immunizations (FIGS. 1A-1B). ELISAresponses were comparable to the homologous and heterologous gp140strains (FIGS. 1A-1B).

Neutralizing Antibody Responses Elicited by Clade A and Clade C Trimers

To assess the neutralization profile afforded by the stable clade A andclade C trimers, TZM.b1 assays (Li et al., Supra; Montefiori et al.,Supra) were performed using a panel of tier 1 and tier 2 viruses with abroad range of neutralization sensitivities from clades A, B and C. Thecriteria for was defined as: (i) >3-fold above pre-immune background,(ii) >2-fold above a concurrent murine leukemia virus (MuLv) control,and (iii) an absolute ID₅₀ titer>60. Guinea pigs immunized with eitherclade A or clade C trimers developed robust, cross-clade neutralizingactivity against neutralization sensitive tier 1 clade A, B and Cviruses (DJ263.8, SF162.LS and MW965.26 respectively) with ID₅₀ titersagainst MW965.26 ranging from 14,274 to 33,847 (FIG. 6). Noneutralization was observed against the autologous vaccine strains.Without intending to be bound by scientific theory, this resultpresumably reflected their inherent neutralization resistant phenotypes.

NAb responses were assessed against more stringent tier 2 primaryisolate viruses. Lower titer but reproducible neutralization activityagainst select tier 2 clade A, B and C viruses was detected in sera fromanimals following immunization (FIG. 6). The magnitude and consistencyof responses against tier 2 viruses were substantially lower thanagainst tier 1 viruses. Nevertheless, a degree of tier 2 neutralizationactivity was consistently observed. Furthermore, the clade C trimerelicited responses of increased magnitude and breadth compared with theclade A trimer. A graphical summary of NAb titers against tier 2 virusesin pre- and post-immunization sera is shown in FIG. 2. Overall, theclade C trimer elicited detectable NAb responses against 27%, 20% and47% of tier 2 viruses tested from clades A, B and C, respectively,whereas the clade A trimer elicited detectable NAb responses against13%, 7% and 27% of tier 2 viruses tested from clades A, B and C,respectively (FIG. 7). These data demonstrate the immunogenicity ofthese stable clade A and clade C trimers and show the utility of thispanel of viruses for providing a systematic tiered approach forassessing NAb responses elicited by HIV-I Env immunogens.

Neutralizing Antibody Responses of Purified IgG

To confirm the results of the NAb assays using serum, additionalneutralization assays were performed using IgG purified from serum ofimmunized animals. Purified IgG demonstrated potent neutralizingactivity against the panel of tier I viruses at low concentrations(0.1-0.4 μg/ml for MW965.26) as well as detectable neutralizationactivity against a limited number of tier 2 viruses includingZMI97M.PB7, ZMIO9F.PB4, 0439.v5.ci and 6535.3 (FIG. 8). Sample raw virusneutralization data using purified IgG against the clade C tier 2 virusZMIO9F.PB4 is shown in FIG. 3. Control IgG from naive sera exhibited noneutralization activity.

Variable Loop Peptide Responses

Antibody responses against V1, V2 and V3 peptides from clade A 92UG037.8and clade C CZA97.012 gp140 were assessed. A scrambled 92UG037.8 V3peptide was utilized as a negative control. Sera from guinea pigsimmunized with both clade A and clade C trimers exhibited ELISAresponses against linear V3 loop peptides but not against the scrambledpeptide (FIGS. 4A-4B). These responses were comparable againsthomologous and heterologous V3 sequences, and lower ELISA titers wereobserved against V1 and V2 peptides. V3 peptide competitionneutralization assays were performed from representative animals thatreceived the clade A (guinea pig #5) and clade C (guinea pig #10)trimers. Neutralizing activity against select tier 1 and tier 2 viruseswas partially blocked by both homologous and heterologous V3 looppeptides but not by the scrambled peptide (FIG. 4C), indicating that theclade A and clade C gp140 trimers elicited NAbs that were directed inpart against conserved elements in the V3 loop. Sequence alignment ofthe V3 loops of 92UG037.8 and CZA97.012 showed substantial sequencehomology (FIG. 4D).

Heterologous Prime/Boost Regimens

DNA priming followed by protein boosting has previously been reported toelicit higher titer antibody responses than either approach alone (Vaineet al. (2008) J Virol. 82:7369; Wang et al. (2006) Virology 350:34).Accordingly, DNA prime, protein boost as well as DNA prime, recombinantadenovirus serotype 26 (rAd26) boost regimens expressing the clade A andclade C trimers were explored. Guinea pigs (n=5/group) were primed with0.5 mg DNA vaccines intramuscularly (i.m.) at weeks 0, 4, and 8 and thenboosted at week 36 with a single immunization of rAd26 vectors or atweeks 36, 40, and 44 with trimer proteins in Ribi adjuvant. High titer,ELISA responses were observed following immunization with both theDNA/protein (FIGS. 5A-5B) and the DNA/rAd26 (FIGS. 5C-5D) regimens. Peaktiters obtained after the three DNA priming immunizations were a meanlog titer of 4.8. Boosting with either a single rAd26 or three purifiedprotein immunizations augmented responses to mean log titers of 6.5,which were comparable to those elicited by the protein only regimen(FIG. 1).

Low levels of tier 1 NAb responses were observed after DNA priming (FIG.9). However, following boosting, the DNA/rAd26 and the DNA/proteinregimens did not induce higher titer tier 1 NAb responses as comparedwith the protein-only regimen. Without intending to be bound byscientific theory, it is though that the DNA vaccines may have eliciteda variety of gp140 protein conformers or oligomers that could haveprimed non-neutralizing antibody responses. These data suggest that, incertain settings, prime/boost vaccine regimens may not necessarily provesuperior to purified protein immunogens.

Discussion

The data presented herein assessed the immunogenicity of highly purifiedCZA97.012 (clade C) and 92UG037.8 (clade A) Env gp140 trimer immunogensthat were selected and engineered for optimal biochemical stability andconformational homogeneity. Most Env trimer immunogens reported to dateare derived from clade B isolates, although recent reports have alsodescribed trimers from other clades (Burke et al., Supra; Kang et al.(2009) Vaccine 37:5120, Epub 2009 Jun. 28). However, the conformationalhomogeneity of those preparations was often not fully assessed. A panelof tier 1 and tier 2 from clades A, B and C viruses with a broad rangeof neutralization sensitivities were utilized to assess virusneutralization. Guinea pigs immunized with clade A and clade C trimersdemonstrated robust, cross-clade neutralizing activity againstneutralization sensitive tier 1 clade A, B and C viruses, as well asclear but low levels of neutralizing activity against select tier 2clade A, B and C viruses. NAb responses against tier 2 isolates wereconfirmed using purified IgG but were substantially lower in magnitudeand less consistent than responses against tier 1 isolates. Antibodyresponses elicited by the trimers were directed in part against the V1,V2 and V3 loops. V3 loop reactivity was observed against bothheterologous viruses, although, without intending to be bound byscientific theory, it is likely that a variety of other epitopes werealso targeted.

Heterologous prime/boost vaccination regimens were also assessed fortheir potential to augment the immunogenicity of the trimer proteinimmunogens. DNA/protein and DNA/rAd26 regimens did not lead to improvedmagnitude or breadth of antibody responses as compared with theprotein-only regimen. In fact, the prime/boost regimens appeared toelicit lower NAb responses despite comparable ELISA binding antibodyresponses. These finding contrast with previous reports highlighting theimproved humoral responses obtained with DNA/protein or DNA/rAd regimensas compared to protein-only regimens in other systems (Seaman et al.(2005) J. Virol. 79:2956; Vaine et al., Supra; Wang et al. (2005) J.Virol. 79:7933; Wang et al., Supra). It is hypothesized that the lowerNAb activity observed in prime/boost regimens in the present study mayhave been related to a heterogeneous mixture of Env conformers oroligomers expressed by DNA vaccines, which could have skewed theantibody responses towards non-neutralizing epitopes. Taken together,these findings indicate that the optimal regimen may be dependant on theparticular antigen or system utilized.

In summary, the results described herein demonstrate the immunogenicityof clade A and clade C trimers that have been selected and engineeredfor optimal purity and stability. Importantly, the panel of tier 1 andtier 2 viruses from clades A, B, and C allows a rapid assessment of NAbprofiles against a diversity of viruses and may prove useful forcomparative immunogenicity studies of novel candidate HIV-1 Envimmunogens.

Example II Materials and Methods

HIV-1 gp140 Trimers

92UG037.8 (clade A) and CZA97.012 (clade C) gp140 trimers with aC-terminal T4 bacteriophage fibritin trimerization domain (foldon) andpoly-histidine motif were expressed in insect cells using the Bac-to-Bacsystem (Invitrogen) as previously described (Chen et al. (2000) J Biol.Chem. 275:34946; Frey et al., Supra). Briefly, recombinant baculoviruswas generated according to the manufacturer's protocol and amplified inSf9 insect cells. For large-scale production, 12 liters of Trichoplusiani (Hi-5) cells (2×10⁶ cells/ml) were infected at the optimalmultiplicity of infection. The supernatant was harvested 68 hours postinfection by centrifugation and concentrated to 2 liters, followed byimmediately exchanging into phosphate buffered saline (PBS) in atangential flow filtration system, ProFlux M 12 (Millipore). After aclarifying spin and adding imidazole to the final concentration of 15mM, the supernatant was loaded onto a nickel column at a flow rate of 1ml/min, then washed with 15 mM imidazole in PBS, followed by furthersequential washes with 40 mM and 60 mM imidazole in PBS. The protein waseluted with 300 mM imidazole in PBS. The fractions containing thepurified protein were pooled, concentrated, and further purified by gelfiltration chromatography on Superose 6 (GE Healthcare). The protein wasconcentrated, frozen in liquid nitrogen, and stored at −80° C.

DNA Vaccines

Human codon optimized gene sequences for the clade C and clade A gp140trimers with a C-terminal T4 bacteriophage fibritin trimerization domain(Bower et al. (2004) J. Virol. 78:4710; Yang et al. (2002) J. Virol.76:4634) and poly-histidine motif were synthesized commercially(Geneart) and cloned into the Sali-BamHI restriction sites of a pCMVeukaryotic expression vector. Gene inserts were verified by diagnosticrestriction digests, DNA sequencing and expression testing in 293 cells.Endo-toxin free preparations of pCMV-CZA97012-gp140 andpCMV-92UG037-gp140 (Qiagen) were utilized for immunization protocols.

Recombinant Adenovirus Serotype 26 Vectors

Replication-incompetent, E1/E3-deleted recombinant adenovirus serotype26 (rAd26) vectors expressing clade A and clade C gp140 trimers with aC-terminal T4 bacteriophage fibritin trimerization foldon domain (Boweret al., Supra; Yang et al., Supra) and poly-histidine motif wereprepared as previously described (Abbink et al. (2007) J. Virol.81:4654).

Animals and Immunizations

Outbred female Hartley guinea pigs (Elm Hill) were housed at the AnimalResearch Facility of Beth Israel Deaconess Medical Center under approvedInstitutional Animal Care and Use Committee (IACUC) protocols. Proteintrimer, (100 μg/animal) were administered at 4 or 5 week intervals in500 μl PBS in Ribi adjuvant (Sigma) at three sites: 2 subcutaneous (sq)(200 μl/site) and 1 intraperitoneal (i.p.) (100 μl/site). Endo-toxinfree DNA vaccines (500 μg/animal) were administered intramuscularly in500 μl PBS divided between the right and left quadriceps at 4-weekintervals. Recombinant Ad26 vectors (5×10¹⁰ vp/animal) were administeredintramuscularly in 500 μl saline divided between the right and leftquadriceps. Serum samples were obtained in anesthetized animals from thevena cava.

ELISA

Serum binding antibody titers against gp140 trimers and linear peptides(New England Peptide) were determined by an end point ELISAs. Ninety-sixwell maxisorp ELISA plates (Thermo Fisher Scientific) coated overnightwith 100 μl/well of 1 μg/ml clade A gp140, Clade C gp140 or V1-V3 linearpeptide loops in PBS were blocked for 3 hours with PBS containing 2% BSA(Sigma) and 0.05% Tween 20 (Sigma). Guinea pig sera were then added inserial dilutions and incubated for 1 hour at room temperature. Theplates were washed three times with PBS containing 0.05% Tween 20 andincubated for 1 hour with a 1/2000 dilution of a HRP-conjugated goatanti-guinea pig secondary antibody (Jackson ImmunoResearchLaboratories). The plates were washed three times and developed withSureBlue TMB microwell peroxidase (KPL Research Products), stopped byaddition of TMB stop solution (KPL Research products) and analyzed atdual wavelengths 450 nm/550 nm on a Spectramax Plus ELISA plate reader(Molecular Devices) using Softmax Pro 4.7.1 software. ELISA end pointtiters were defined as the highest reciprocal serum dilution thatyielded absorbance>2-fold background.

TZM.b1 Neutralization Assay

NAb responses against tier 1 and tier 2 HIV-1 pseudoviruses weremeasured using a luciferase-based assay in TZM.b1 cells as previously(Li et al., Supra; Montefiori et al., Supra). This assay measured thereduction in luciferase reporter gene expression in TZM-b1 cellsfollowing a single round of virus infection. The IC₅₀ was calculated asthe concentration that caused a 50% reduction in relative luminescenceunits compared with the virus control wells after the subtraction ofcell control relative luminescence units. Briefly, 3-fold serialdilutions of serum samples were performed in triplicate (96-well flatbottom plate) in 10% DMEM growth medium (100 μl/well). 200 TCID50 ofvirus was added to each well in a volume of 50 μl and the plates wereincubated for 1 hour at 37° C. TZM.b1 cells were then added (1×10⁴/wellin 100 μl volume) in 10% DMEM growth medium containing DEAE-Dextran(Sigma) at a final concentration of 11 μg/ml. Murine leukemia virus(MuLV) negative controls were included in all assays to rule outnon-specific inhibition. Confirmatory assays were performed utilizingIgG purified by immobilized protein A columns (Pierce). To test variableloop peptide reactivity, purified IgG samples was incubated with V1, V2,or V3 linear peptides for 1 hour at 37° C. prior to addition ofpseudovirus. A negative control scrambled 92UG037 V3 peptide was alsoincluded in these assays. Viruses in the tier 1 panel were: MW965.26(clade C), DJ123.8 (clade A), SF162.LS (clade B), BaL.26 (clade B),92UG037.8 (clade A) and CZA97.012 (clade C). Viruses in the tier 2 cladeA panel were: Q769.d22, Q168.a2, Q842.d12, 3718.v3, 0330.v4 and 0439.v5.Viruses in the tier 2 clade B panel were: WIT04160.33, AC10.0.29,REJ0451, 6535.3, SC422661 and TRO.11. Viruses in the tier 2 clade Cpanel were: ZM109F.PB4, ZM249M, CAP45.2, Du123.6, Du422.1, and ZM197M.

Gene Structure and Cloning Enzymes

5′ unique cloning enzyme: SaD; Kozak sequence: GCCACC; 3′ unique cloningenzyme: BamHI; Gene structure: Sali-GCCACC-ATG..Stop-BamHI; Optimize:YES. The 92UG037 gp140-6× His trimer (MCONS leader) amino acid sequenceis depicted in FIG. 12A. The CZA97.012 gp140-6× His trimer (MCONSleader) amino acid sequence is depicted in FIG. 12B.

REFERENCES

Beddows et al. (2007) Virology 360:329

Li et al. (2006) J Virol. 80:1414

Bower et al. (2006) Vaccine 24:5442

Kim et al. (2005) AIDS Res. Hum. Retroviruses 21:58

Frey et al. (2008) Proc. Natl. Acad. Sci. USA 105:3739

Barouch (2008) Nature 455:613

Beddows et al. (2007) Virology 360:329

Berger et al. (1999) Annu. Rev. Immunol. 17:657

Berman et al. (2002) Aids 8:591

Binley et al. (2000) J Virol. 74:627

Bower et al. (2006) Vaccine 24:5442

Burton et al. (2004) Nat. Immunol. 5:233

Carrow et al., (1991) AIDS Res. Hum. Retroviruses 7:831

Crooks et al. (2007) Virology 366:245

Derby et al. (2007) Virology 366:433

Dey et al. (2007) J Virol. 81:5579

Flynn et al. (2005) J Infect. Dis. 191:654

Gallo et al. (2003) Biochim. Biophys. Acta 1614:36

Kim et al. (2005) AIDS Res. Hum. Retroviruses 21:58

Li et al. (2006) J Virol. 80:1414

Montefiori et al. (2007) PLoS Med. 4:e348

Momer et al. (2009) J Virol. 83:540

Nara et al. (1988) J Virol. 62:2622

Page et al. (1991) Vaccine 9:47

Pantophle and Burton (2006) Annu. Rev. Immunol. 24:739

Pitisuttithum et al. (2006) J Infect. Dis. 194:1661

Scheid et al. (2009) Nature 458:636

Vogel et al. (1994) J Immunol. 153:1895

Walker et al. (2009) Science 326:285

Wyatt et al. (1998) Nature 393:705

Zhang et al. (2001) J Biol. Chem. 276:39577

Zolla-Pazner et al. (2008) Virology 372:233.

What is claimed:
 1. An isolated nucleic acid molecule encoding at leastone gp140 polypeptide, wherein said gp140 polypeptide comprises an aminoacid sequence at least 98% identical to residues 30-708 of SEQ ID NO:2,and wherein said gp140 polypeptide comprises an oligomerization domain.2. An isolated nucleic acid molecule encoding at least one gp140polypeptide, wherein said gp140 polypeptide comprises an amino acidsequence at least 98% identical to SEQ ID NO:2, and wherein said gp140polypeptide comprises an oligomerization domain.
 3. The isolated nucleicacid molecule of claim 1, wherein said amino acid sequence comprises SEQID NO:8.
 4. The isolated nucleic acid molecule of claim 1, wherein saidamino acid sequence comprises SEQ ID NO:6.
 5. The isolated nucleic acidmolecule of claim 3, wherein said amino acid sequence comprises SEQ IDNO:6.
 6. The isolated nucleic acid molecule of claim 1, wherein saidoligomerization domain is a T4 fibritin trimerization domain.
 7. Avector comprising the isolated nucleic acid molecule of claim
 1. 8. Thevector of claim 7, wherein said vector is a plasmid.
 9. The vector ofclaim 7, further comprising a promoter that drives expression of saidgp140 polypeptide.
 10. A method of recombinantly producing a gp140polypeptide, said method comprising the steps of: (a) contacting a cellwith the vector of claim 7; (b) expressing said at least one gp140polypeptide; and (c) purifying said gp140 polypeptide.
 11. The method ofclaim 10, wherein the expressed gp140 polypeptide forms a stabilizedtrimer.
 12. The method of claim 10, wherein said cell is a bacterialcell, a yeast cell, an insect cell, or a mammalian cell.
 13. The methodof claim 12, wherein said cell is a mammalian cell.
 14. A pharmaceuticalcomposition comprising the recombinantly produced gp140 polypeptide ofclaim 10 and a pharmaceutically acceptable carrier, excipient, ordiluent.
 15. The isolated nucleic acid molecule of claim 2, wherein saidoligomerization domain is a T4 fibritin trimerization domain.
 16. Avector comprising the isolated nucleic acid molecule of claim
 2. 17. Avector comprising the isolated nucleic acid molecule of claim
 6. 18. Avector comprising the isolated nucleic acid molecule of claim
 15. 19. Amethod of recombinantly producing a gp140 polypeptide, said methodcomprising the steps of: (a) contacting a cell with the vector of claim18; (b) expressing said at least one gp140 polypeptide; and (c)purifying said gp140 polypeptide.
 20. A pharmaceutical compositioncomprising the recombinantly produced gp140 polypeptide of claim 19 anda pharmaceutically acceptable carrier, excipient, or diluent.
 21. Theisolated nucleic acid molecule of claim 2, wherein said gp140polypeptide comprises the amino acid sequence of SEQ ID NO:2.
 22. Themethod of claim 19, wherein the expressed gp140 polypeptide forms astabilized trimer.